title: CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand.
abstract: The CRISPR/Cas9 system has attracted significant attention for its potential to transform genome engineering. We and others have recently shown that the RNA-guided Cas9 nuclease can be employed to engineer the Drosophila genome, and that these modifications are efficiently transmitted through the germline. A single targeting RNA can guide Cas9 to a specific genomic sequence where it induces double-strand breaks that, when imperfectly repaired, yield mutations. We have also demonstrated that 2 targeting RNAs can be used to generate large defined deletions and that Cas9 can catalyze gene replacement by homologous recombination. Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have shown similar promise in Drosophila. However, the ease of producing targeting RNAs over the generation of unique sequence-directed nucleases to guide site-specific modifications makes the CRISPR/Cas9 system an appealingly accessible method for genome editing. From the initial planning stages, engineered flies can be obtained within a month. Here we highlight the variety of genome modifications facilitated by the CRISPR/Cas9 system along with key considerations for starting your own CRISPR genome engineering project.
authors.affiliation: Genetics Training Program; University of Wisconsin-Madison; Madison, WI USA.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Gratz
authors.affiliation: Department of Biochemistry; University of Wisconsin-Madison; Madison, WI USA.
authors.forename: Jill
authors.initials: J
authors.lastname: Wildonger
authors.affiliation: Department of Biomolecular Chemistry; University of Wisconsin School of Medicine and Public Health; Madison, WI USA.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Harrison
authors.affiliation: Genetics Training Program; University of Wisconsin-Madison; Madison, WI USA; Laboratory of Genetics; University of Wisconsin-Madison; Madison, WI USA; Laboratory of Cell and Molecular Biology; University of Wisconsin-Madison; Madison, WI USA.
authors.forename: Kate M
authors.initials: KM
authors.lastname: O'Connor-Giles
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D049750:Genome, Insect; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed
keywords: CRISPR; Cas9; genome engineering; homologous recombination; site-directed mutagenesis